A. G. Yakovlev

Nuclear wavepacket motion producing a reversible charge separation in bacterial reaction centers

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P*, which lead to the fs oscillations in stimulated emission from P* [M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.‐C. Lambry and J.‐L. Martin (1994) Biochemistry 33, 6750–6757] and in QY absorption band of the primary electron acceptor, bacteriochlorophyll monomer BA [A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293–7298] with a set of fundamental frequencies in the range of 10–300 cm−1. We have found that in pheophytin‐modified RCs, the fs oscillations with frequency around 130 cm−1 observed in the P*‐stimulated emission as well as in the BA absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer BA −. These results are discussed in terms of a reversible electron transfer between P* and BA induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+BA −, when a maximal value of the Franck–Condon factor is created.

Coupling of nuclear wavepacket motion and charge separation in bacterial reaction centers

The mechanism of the charge separation and stabilization of separated charges was studied using the femtosecond absorption spectroscopy. It was found that nuclear wavepacket motions on potential energy surface of the excited state of the primary electron donor P* leads to a coherent formation of the charge separated states P+BA −, P+HA − and P+HB − (where BA, HB and HA are the primary and secondary electron acceptors, respectively) in native, pheophytin‐modified and mutant reaction centers (RCs) of Rhodobacter sphaeroides R‐26 and in Chloroflexus aurantiacus RCs. The processes were studied by measurements of coherent oscillations in kinetics at 890 and 935 nm (the stimulated emission bands of P*), at 800 nm (the absorption band of BA) and at 1020 nm (the absorption band of BA −) as well as at 760 nm (the absorption band of HA) and at 750 nm (the absorption band of HB). It was found that wavepacket motion on the 130–150 cm−1 potential surface of P* is accompanied by approaches to the intercrossing region between P* and P+BA − surfaces at 120 and 380 fs delays emitting light at 935 nm (P*) and absorbing light at 1020 nm (P+BA −). In the presence of Tyr M210 (Rb. sphaeroides) or M195 (C. aurantiacus) the stabilization of P+BA − is observed within a few picosseconds in contrast to YM210W. At even earlier delay (∼40 fs) the emission at 895 nm and bleaching at 748 nm are observed in C. aurantiacus RCs showing the wavepacket approach to the intercrossing between the P* and P+HB − surfaces at that time. The 32 cm−1 rotation mode of HOH was found to modulate the electron transfer rate probably due to including of this molecule in polar chain connecting PB and BA and participating in the charge separation. The mechanism of the charge separation and stabilization of separated charges is discussed in terms of the role of nuclear motions, of polar groups connecting P and acceptors and of proton of OH group of TyrM210.

Light control over the size of an antenna unit building block as an efficient strategy for light harvesting in photosynthesis

It was shown that an increase in the bacteriochlorophyll (BChl) c antenna size observed upon lowering growth light intensities led to enhancement of the hyperchromism of the BChl c Qy absorption band of the green photosynthetic bacterium Chloroflexus aurantiacus. With femtosecond difference absorption spectroscopy, it was shown that the amplitude of bleaching of the oligomeric BChl c Qy band (as compared to that for monomeric BChl a) increased with increasing BChl c content in chlorosomes. This BChl c bleaching amplitude was about doubled as the chlorosomal antenna size was about trebled. Both sets of findings clearly show that a unit BChl c aggregate in the chlorosomal antenna is variable in size and governed by the grow light intensity, thus ensuring the high efficiency of energy transfer within the BChl c antenna regardless of its size.

Femtosecond kinetics of electron transfer in the bacteriochlorophyllM‐modified reaction centers from Rhodobacter sphaeroides (R‐26)

Formation of the vibronic wavepacket by 90‐fs excitation of the primary electron donor P in bacteriochlorophyllM‐modified reaction centers is shown to induce nuclear motions accompanied by (1) oscillation of the stimulated emission from excited primary electron donor P∗ and (2) wavepacket motions leading to electron transfer at 293 K from P∗ to bacteriochlorophyll (BL) and then to bacteriopheophytin (HL). The latter motions have low frequency (about 15 cm−1) and are related to protein‐nuclear motions which are along the reaction coordinate. When the wavepacket approaches the intersection of the reactant (P∗BL) and product (P+BL−) potential energy surfaces (∼1.5 ps delay), about 60% of P∗ is converted to the P+BL− state. The P+HL− state formation is delayed by ∼2 ps with respect to that of P+BL−. It is suggested that the wavepacket is transferred to and moves also slowly on the P+BL− potential energy surface and approaches the intersection of the surfaces of P+BL− and P+HL− within ∼2 ps (∼8 cm−1), indicating the electron transfer to HL.

Electron transfer in deuterated reaction centers of Rhodobacter sphaeroides at 90 K according to femtosecond spectroscopy data.

The primary act of charge separation was studied in P+BA– and P+HA– states (P, primary electron donor; BA and HA, primary and secondary electron acceptor) of native reaction centers (RCs) of Rhodobacter sphaeroides R-26 using femtosecond absorption spectroscopy at low (90 K) and room temperature. Coherent oscillations were studied in the kinetics of the stimulated emission band of P* (935 nm), of absorption band of BA– (1020 nm) and of absorption band of HA (760 nm). It was found that in native RCs kept in heavy water (D2O) buffer the isotopic decreasing of basic oscillation frequency 32 cm –1 and its overtones takes place by the same factor ∼1.3 in the 935, 1020, and 760 nm bands in comparison with the samples in ordinary water H2O. This suggests that the femtosecond oscillations in RC kinetics with 32 cm –1 frequency may be caused by rotation of hydrogen-containing groups, in particular the water molecule which may be placed between primary electron donor PB and primary electron acceptor BA. This rotation may appear also as high harmonics up to sixth in the stimulated emission of P*. The rotation of the water molecule may modulate electron transfer from P* to BA. The results allow for tracing of the possible pathway of electron transfer from P* to BA along a chain consisting of polar atoms according to the Brookhaven Protein Data Bank (1PRC): Mg(PB)-N-C-N(His M200)-HOH-O = BA. We assume that the role of 32-cm –1 modulation in electron transfer along this chain consists of a fixation of electron density at BA– during a reversible electron transfer, when populations of P* and P+BA– states are approximately equal.

Exciton dynamics in the chlorosomal antenna of the green bacterium Chloroflexus aurantiacus: experimental and theoretical studies of femtosecond pump-probe spectra

Femtosecond absorption difference spectra were measured for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus at room temperature. Using the relative difference absorption of the oligomeric BChl c and monomeric BChl a bands, the size of a unit BChl c aggregate as well as the exciton coherence size were estimated for the chlorosomal BChl c antenna under study. A quantitative fit of the data was obtained within the framework of the exciton model proposed before [Fetisova et al. (1996) Biophys J 71: 995–1010]. The size of the antenna unit was found to be 24 exciton-coupled BChl c molecules. The anomalously high bleaching value of the oligomeric B740 band with respect to the monomeric B795 band provided the direct evidence for a high degree of exciton delocalization in the chlorosomal B740 BChl c antenna. The effective delocalization size of individual exciton wavefunctions (the thermally averaged inverse participation ratio) in the chlorosomal BChl c antenna is 9.5, whereas the steady-state wavepacket corresponds to the coherence size (the inverse participation ratio of the density matrix) of 7.4 at room temperature.

Dynamic hole burning within special pair absorption band of Rhodobacter sphaeroides (R‐26) reaction center at room temperature

The absorbance spectrum of reaction centers of Rhodobacter sphaeroides at room temperature consists of relatively narrow spectral components which are moving in the femtosecond time scale and can be bleached by femtosecond laser pulses in the short wavelength region with a subsequent broadening and red shift of the bleaching (time constant ∼ 250 fs). These data are discussed in terms of the population of the vibronic wave packets in the ground state by the interaction with phonons at 293K. The motion of these packets is probably responsible for the absorbance spectrum of the primary electron donor P at 293K with enhanced short wavelength components and with suppressed Stokes components.

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of Rhodobacter sphaeroides

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P+BA− in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P+/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P+BA− stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm−1 reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of BA− at 1020 nm is registered in RCs of both mutants. The formation of the BA− band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm−1. Only a partial stabilization of the P+BA− state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P+ and BA− is proposed to explain rapid stabilization of the P+BA− state in native RCs. Small oscillatory components at ∼330–380 cm−1 in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P+BA− free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P+ and BA− is assumed to be another potential contributor to the mechanism of P+BA− stabilization.

Primary Processes of Charge Separation in Reaction Centers of YM210L/FM197Y and YM210L Mutants of Rhodobacter sphaeroides

Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll BA− at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* → P+BA− and to the absence of stabilization of separated charges in the state P+BA−. Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule PB of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm−1 in the single mutant YM210L to ∼100 cm−1 in the double mutant. Oscillations with 100–150 cm−1 frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P+BA− state of both mutants reflect a motion of the PB molecule relatively to PA in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the BA− absorption band are conserved within a shorter time ∼0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.

Modeling of reversible charge separation in reaction centers of photosynthesis: an incoherent approach.

Primary charge separation in reaction centers (RCs) of bacterial photosynthesis is modeled in this work. An incoherent population dynamics of RCs states is formulated by kinetic equations. It is assumed that charge separation is accompanied by regular motion of the system along additional coordinates. This motion modulates an energetics of the reactions, and this modulation causes femtosecond oscillations in the population of the states. The best qualitative and quantitative accordance with experimental data on native, modified and mutant RCs of Rba. sphaeroides is achieved in the five states model that includes two excited states P(*)905BAHA and P(*)940BAHA and three charge separated states I, P(+)BA(-)HA and P(+)BAHA(-) (P is a primary electron donor, bacteriochlorophyll dimer, BA and HA are electron acceptors, monomeric bacteriochlorophyll and bacteriopheophytin in active A-branch respectively). The excited states emit at 905 and 940 nm and have approximately the same energy and high interaction rate. The intermediate state I is populated earlier than the P(+)BA(-)HA state and has energy close to the energy of the excited states, a high rate of population and depopulation and spectral identity to the BA(-). A sum of the I and P(+)BA(-)HA populations fits the experimental kinetics of the BA(-) absorption band at 1020 nm. The model explains an oscillatory phenomenon in the kinetics of the P(*) stimulated emission and of the BA(-) absorption. In the schemes without the I state, accordance with the experiment is achieved at unreal parameter values or is not achieved at all. A qualitative agreement of the model with the experiment can be achieved at a wide range of parameter values. The nature of the states I and P(*)940BAHA is discussed in terms of partial charge separation between P and BA and inside P respectively.